反基因锁核酸体外抑制乙肝病毒前S1基因表达

摘要

Objective To investigate the inhibitory effects of hepatitis B virus(HBV) preS1 gene-specific anti-gene locked nucleic acid(LNA) on HBV replication and expression in HepG2.2.15 cells.Methods The anti-gene LNA which were complementary to the purine rich region of HBV preS1 gene were synthesized and transfected by cationic liposomes into HepG2 2.2.15 cells.The HBsAg,preS1-Ag and HBV DNA of supernatant was tested by time-resolved fluorescence immune assay(TRFIA),real-time fluorescent quantitative PCR (FQ-PCR) and enzyme linked immunosorbent assays(ELISA) at 1,3,5 and 7 d after treatment.LNA's cyto-toxicity on cell was evaluated by MTT method.Results The anti-gene LNA that targeting on the purine rich region of HBV preS1 gene showed strong inhibitory effects on replication of HBV DNA and the expression of HBsAg and preS1-Ag with the inhibition rates of 65.99％,67.49％ and 63.88％ respectively after 7 days.There's no obvious toxicity on cell.Conclusions Anti-gene locked nucleic acid(LNA) that targeting on the purine rich region of HBV preS1 gene has show strong inhibition on HBV in vitro.It has a therapeutic potential in the treatment of patients infected with HBV.%目的 探讨针对乙肝病毒前S1基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用.方法 针对乙肝病毒前S1基因同聚嘌呤区,利用RNA structure软件分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术(FQ-PCR)、时间分辨免疫荧光技术(TRFIA)和酶联免疫法(ELISA)分别监测1、3、5和7d细胞培养上清液中HBV DNA、HBsAg和前S1抗原的含量；四甲基偶氮唑蓝(MTT)法检测锁核酸对细胞代谢的影响.结果 加入锁核酸后,对HBV DNA复制、HBsAg和前S1抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7d后抑制率分别达65.99％、67.49％和63.88％.LNA对细胞代谢无明显影响.结论 针对乙肝病毒前S1基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据.